INTERFERON GLYCOPROTEIN

Abstract: An interferon glycoprotein of neuronal origin (N-IFN- gamma ) which has a molecular weight of from about 50 to about 70 kDa, is capable of interaction with at least two monoclonals directed against spatially distinct epitopes of lymphocyte-derived IFN- gamma , exerts parasite-stimulating and anti-viral actions, and is capable of induction of MHC and of enhancing muscle cell proliferation; a pharmaceutical composition comprising such glycoprotein; a process for preparing such interferon glycoprotein; and methods of performing interferon therapy on mammals including man.

Description: Interferon glycoprotein Technical field The present invention relates to interferon proteins and, more particularly, it relates to an interferon glyco¬ protein of neuronal origin (N-IFN-2)- The invention fur¬ ther relates to a pharmaceutical composition comprising such glycoprotein, and a process for preparing such inter¬ feron glycoprotein. Finally, the invention involves a me- thod of performing .interferon therapy, such as antiviral therapy, on mammals including man. Background art Interferons (IFNs) are proteins with antiviral, iπunu- nomodulatory, and antiproliferative activities produced by mammalian cells in response to a variety of inducers. IFNs are classified into three groups, , β and 2J IFN-Ϊ, a glycoprotein with a molecular weight ranging from 14 to 25 kD, is previously described to be produced only by activa- τed lymphoid cells. It acts as a ci er under physiological conditions and its effects include activation of macropha- ges, induction of major histocompatibility antigens (MKC), T-cell homing, regulation cf cell proliferation and inter¬ ference with virus replication. Another remarkable effect of IFN-y is that it stimulates proliferation of Trypano- soma brucei brucei (T.b.b. ). R. Kiefer et al. ( euroscience Vol 45, No. 3, pp 551- 560, 1991) describe a ϊ-interferon-like immunoreactive mo¬ lecule derived from εubpopυlations cf rat neurons. This molecule hes been further characterized in the article, such as by giving an approximate molecular weight and by stating immunoreactivity with monoclonal antibody DBl di¬ rected against rat-7-interferon. However, further charac¬ terizing features of this molecule such as non-glycosyle- tion, non-involvment in the control of major histocompati¬ bility complex expression on glial cells, and non-immuno- reactivity with other monoclonal antibodies directed against rat-y-interferon, constitute a clear indication that the molecule disclosed by Kiefer et al. is in fact not an interferon-2-immunoreactive molecule. Thus, all characterizing features disclosed in the article and taken together point to the fact that Kiefer et al. were con¬ fronted with an artifact that happened to have an approxi¬ mate molecular weight of 60000 and was non-specifically immunoreactive with monoclonal antibody DB1. Furthermore, it is important to note that Kiefer et al. have made no disclosure whatsoever of any isolation of their material. Summary of the invention A principal object of the present invention is to provide an interferon glycoprotein of neuronal origin, said glycoprotein being referred to herein as N-IFN-7. Another object of the present invention is to provide a pharmaceutical composition comprising said glycoprotein in an effective amount. Yet another object of the invention is to provide a process for preparing such an interferon glycoprotein of neuronal origin. Still further objects of the invention are to provide methods of performing interferon therapy on mammals inclu¬ ding man, such as antiviral therapy. These and other objects of the invention are obtained by an interferon glycoprotein of neuronal origin which has a molecular weight of from about 50 to about 70 kDa, is capable of interaction with at least two monoclonals di¬ rected against spatially distinct epitopes of lymphocyte- derived IFN-2, exerts parasite-stimulating and anti-viral actions, and is capable of induction of MHC and of enhan¬ cing muscle cell proliferation. According to a preferred aspect of the invention the glycoprotein displays on SDS-PAGE and silver staining three distinct bands corresponding to approximate molecu¬ lar weights of 66, 62 and 54 kDa. The present invention also provides for a pharmaceu¬ tical composition comprising an effective amount of said glycoprotein in admixture with a pharmaceutically accept¬ able vehicle or carrier. The invention also provides for a process for prepa¬ ring an interferon glycoprotein as disclosed above, said process comprising the following, steps: a) providing a starting material originating from neurons of peripheral sensory ganglia; b) preparing a homogenate in suspension of the mate¬ rial from step a); c) passing the suspension resulting from step b) through a column having immobilized therein a first mouse monoclonal anti-rat IFN-2 antibody and eluting the desired material using an alkaline buffer solution; d) passing the eluate from step c) through another column having immobilized therein a second mouse monoclo¬ nal anti-rat IFN-2 antibody and repeating the elution pro¬ cedure of step cJ; and e) recovering the eluate from step d) comprising the desired glycoprotein. Accordingly, the antibodies are directed against the spatially distinct epitopes of lymphocyte-derived IFN-2, and are preferably selected from DB1 and DB16. It is preferred that the elution procedure of steps c) and d) above is performed with a buffer solution having a strong alkaline pH, such as a pH of about 12. Additionally, the present invention provides methods of performing interferon therapy on mammals including man, said methods comprising administering therapeutically effective amount of the glycoprotein or the composition as defined above. Among such methods said therapy may reside in the treatment of a viral infection, in the provision of cell growth regulation, or in the regulation of the production of a cell-produced substance. According to a particularly preferred method it is directed to antiviral therapy comprising the administra¬ tion of an amount capable of inhibition of a viral infec¬ tion of the polypeptide or the composition as defined above to a mammal subject to such therapy. The invention will be further illustrated in the fol¬ lowing by non-limiting examples. In these examples refe¬ rence is made to appended drawings. Brief description of the drawings Figure 1 shows the effects of DB1 column eluted N- IFN-2 and rIFN-2 on trypanosome growth; Figure 2 illustrates evaluation of N-IFN-Z purifi¬ cation from trigeminal ganglion homogenates by measuring 3 H-thymidine incorporation by T.b.b.; Figure 3 shows SDS-PAGE and silver staining of N-IFN- 2 obtained after consecutive elutions from DB1 and DB16 columns; 3 Figure 4 illustrates growth stimulation through H- thymidine uptake in trypanosome cultures; Figure 5 illustrates N-IFN-2 induced MHC class I (A) and II (B) antigens on macrophages; Figure 6 shows N-IFN-2 induced MHC class I (A) and II (B) induction on skeletal muscle cells in a cell-ELISA system; and Figure 7 shows a diagram on proliferation index as a function of exposure time. Examples illustrating the present invention EXAMPLE 1 Purification of the neuronal IFN-2 like iππnunoreactive molecule Trigeminal ganglia were collected from Sprague-Dawley rats and pestled in liquid nitrogen. The material was sus¬ pended to obtain a 5% homogenate in 10 mM phosphate buffe¬ red saline (PBS), 1 mM EDTA and 0.1 mM phenyl ethylsulfo- nylfluoride (PMSF) (Sigma, St. Louis, USA) at pH 7.4. The homogenate was sonicated for 2 min on ice using a Branson sonifier (cell disruptor B15, output 4, 40% duty cycle). The suspension was centrifuged on a Beckman GPR centrifuge at 3,800 rp for 20 min and the supernatant used for anti¬ body affinity purification of N-IFN-2. Two different mouse monoclonal anti-rat IFN-2T antibo¬ dies, DB1 and DB16, were used for antibody affinity co¬ lumns. Both antibodies had been purified from mouse asci- tes fluid on protein A [van der Meide, P.H., Borman, A.H., Beijaars, H.G., Dubbeld, M.A., Botman, C.A.D. and Schellekens, H. Ly phokine Res. 1989, 8, 439]. DB1 is an IgGl antibody binding to both rat and mouse IFN-2. DBl neutralizes activities of IFN-2 in vivo and in vitro with respect to both MHC inducing and antiviral effects. DBl6 is an IgG2A antibody, which binds to rat, but not to mouse IFN-2- Competition experiments have shown that DBl6 binds to a different epitope of IFN-y than DBl. DBl6 does not, or only slightly, neutralize antiviral activity of IFN-tf. The antibodies were immobilized to cyanide bromide activa¬ ted sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the manufacturers' instructions to obtain 3 mg antibody/ml gel. To remove still remaining larger sized particles, a precolumn, Sepharose CL6B (Pharmacia), was used. Equili¬ bration of the column was made with 10 mM phosphate buffe¬ red saline (PBS), pH 7.4. The size of the DBl-column was 1.5x2.0 cm and the flow rate 0.1 ml/min. Washing was made with 10 mM PBS, pH 7.4, containing 0.5 M NaCl and elution of the sample was performed with 0.2 M potassium phosphate buffer, pH 12, containing 0.5 M NaCl. The eluate was neut¬ ralized with 1 M KH2P04 buffer/eluate in a ratio of 1:5. The sample, still in high salt solution, was then subjec¬ ted to a similar DBl6 column and the procedure repeated. The sample was sterile filtered directly after elution and kept at 4°C until use. The purification steps were assayed by analysing the capacity of the various fractions to sti- mulate growth of T.b.b. (see below). EXAMPLE 2 Electrophoretic characterization of the purified IFN-tf like molecule Protein concentration of the alkali eluted N-IF -2 was measured according to Bradford [Bradford, M.M. Analy¬ tical Biochemistry 1976, 72, 248]. Protein concentration ranged from 1 to 3 μg/ml buffer in the different purifi- cations. Polyacrylamide gel elektrophoresis (PAGE, Bio-Rad, USA) under reducing (sodium dodecyl sulphate + 2-mercapto- ethanol) conditions was performed according to Laem li [Laemmli, U.K. Nature 1970, 227, 680] and molecular weight standards run in parallel. Proteins were silver stained (Bio-Rad) [Merril, C.R., Goldman, D., Sedman, S.A. and Ebert, M.H. Science 1981, 211, 1437]. To study if protein stained on gel after PAGE correlated with N-IFN-2 bioacti- vity, PAGE was run also under non-denaturing and non-redu- cing conditions, one strip of the gel was silver stained and one strip cut with a razor blade into 17 pieces. Each of them was homogenized into 1 ml of PBS with a syringe, gel material spun down, and supernatant assayed for T.b.b. growth stimulation (see below). EXAMPLE 3 Trypanosome growth stimulation A variable antigen type An Tat 1/1, derived from a stabilate EATRO 1125, of T.b.b. (obtained from Dr. Nestor van Meirvenne, Laboratory of Serology, Institute of Tropi¬ cal Medicine Prins Leopold, Antwerp, Belgium) was passaged in Sprague-Dawley rats. Trypanso es were purified from the blood of these rats as described by Lanham and Godfrey [Lanham, S.M. and Godfrey, D.G. Exp.Parasitol. 1070, 28, 521] . 200 μl aliquots of mobile parasites at concentra- 5 6 7 tions of 10 , 10 and 10 trypansomes/ml culture medium were added to round-bottomed 96-well microtitre plates (Nunclon, Nunc, Denmark). To these wells 10 μl aliquots of the purified N-IFN-2 in serial dilutions were added or of 3 recombinant IFN-2, 10 units/ml [van der Meide, P.H., Dubbeld, M., Vijverberg, K. , Kos, T., Schellekens, H.J. Gen.Virol. 1986, 67, 1059], while control wells were cultured with medium alone. The trypanosomes were counted after 4 h and 8 h in culture. The content of each well was mixed 10 times with a 100 μl micropipette. Ten (10) μl of the fluid were added to 90 μl of a trypan blue solution followed by thorough mixing. Bϋrker chambers were used to count the number of mobile trypansomes that excluded try¬ pan blue at x200 magnification. The density of living try¬ panosomes per ml medium present in the wells was calcula¬ ted. The proliferation of trypanosomes was also estimated by a thymidine incorporation assay. Purified T.b.b. were cultivated in the presence or absence of the N-IFN-2 mole- 3 cule or recombinant IFN-y. H-methyl hymidine (10 μl, 100 μCl/ml; Amersham, Little Chalfont, UK) was added to the wells as the start of the culture. At 4 or 8 h after pla- ting, trypansomes were harvested and thymidine incorpora¬ tion counted. EXAMPLE 4 MHC class I and II antigen induction on macrophages Sprague-Dawley rats were injected intraperitoneally with 2 ml of 3% thioglycollate (Sigma) as described by Kraemer et al [Kraemer, Y.I., Lopez, R.D. and Reaven, G.M. J.Clin.Endocrinol.Metab. 1985, 61, 335]. Four days post injection, the peritoneal cavities were rinsed for 3 min with complete Iscove's medium (Flow Laboratory, Irvine, U.K. ) supplemented with 1% (v/v) minimum essential medium (Flow), 50 IU/ml penicillin, 60 μg/ml streptomycin (Gibco, Paisley, U.K.), 2 mM glutamine (Flow), and 1% normal rat serum. After 3 washes in the same medium, the cell number of the suspension was adjusted to 1.1 - 1.5 x 10 viable cells/ml medium. For assessment of MHC class I and II expression, a cell-ELISA was used. 100 μl aliquots of peritoneal cells were poured into flat-bottomed 96-well polystyrene micro- titre plates (Nunc, Copenhagen, Denmark). After 2.5 h of incubation at 37°C and 5% CO,, the medium was gently dis¬ carded. Cells other than macrophages are thereby removed, while macrophages remain adhesive to the plastic surface. Subsequently, the wells were refilled with medium (200 μl/well) and incubated for 72 h at 37°C and 5% Co 2 , provi- ding a confluent cell layer in the wells. The medium was then discarded and the wells filled with either 200 μl me¬ dium alone, or medium containing purified N-IFN-tf at dif¬ ferent dilutions or recombinant rat IFN-2C at concentra¬ tions of 1.0 U/ml and 10 U/ml. Subsequently, the medium was discarded and the cells were fixed in 95% ethanol (-20°C) for 30 sec. The plates were washed 4 times in PBS, pH 7,4, followed by the addi¬ tion of 50 μl aliquots of 2% normal horse serum (Vector Lab., Burlingame, CA, USA), dissolved in PBS and incubated for 30 min at room temperature to block non-specific bin¬ ding sites on the microtitre plates. After discarding the horse serum, monoclonal mouse anti-rat class I antibody (0x18) (Serotec, Kidlington, Oxford, U.K.), diluted 1:500 in 2% normal horse serum or anti-rat class II antibody (0x6) (Serotec) diluted to a final concentration of 3 μg/ml in 2% normal horse serum were added and incubated overnight at 4°C. After 4 washings with PBS, biotinylated horse anti-mouse IgG (Vector) diluted 1:80 in 1% normal rat serum was added for 1 h at room temperature followed by avidin-biotin alkaline phosphatase complex (ABC-AP, Vector) diluted 1:100 in PBS for 30 min. Unbound ABC-AP was removed by 4 consecutive washings with PBS, and 100 μl/well of freshly-prepared enzyme substrate solution-was added. The preparation was formed by dissolving 3 tablets of p-nitrophenyl phosphate (Sigma) in a solution of 9.7 ml diethanolamin, 0.02 MgCl 2 x 6 H 2 0, 10 ml 1 M HCI and 70 ml distilled water. Absorbance was measured every 5 min for 30 min at 405 nm in a photometer (Labsystems Multiskan mcc/340; Labsystem, Helsinki, Finland). EXAMPLE 5 Antiviral bioassay The antiviral bioactivity of the neuronal IFN-y pre¬ paration was determined by inhibiton of the cytopathic effect (CPE) of vesicular stomatitis (VSV) on onolayers of a rat continuous cell line (Ratec) sensitive to IFN-2, -β and -a. Ratec cells (3.5x10 cells/ml; 100 μl/well) were grown to confluency in the wells of a 96-well micro- titer plate in Dulbecco's minimal essential medium (DMEM) containing 10% fetal calf serum (FCS) and antibiotics. Confluency was reached after an overnight incubation at 37°C in a humidified atmosphere containing 7% Co 2 - Samples were then applied in serial twofold dilutions in DMEM with 1% FCS and antibiotics. Each sample was run in duplicate and assayed at least twice in two independent assays. In each series of determinations, eight wells served as a cell control and eight wells as a virus control. A labora¬ tory standard preparation of recombinant IFN-2 was also titrated on the plate. Samples, standard and controls were incubated for 20 h at 37 C C, after which the supernatant fluid was removed and replaced by 100 μl of a suspension of VSV in DMEM containing 1% FCS. The virus concentration was chosen in such a way that about 90% of CPE was obtai¬ ned after 24 h of incubation. After challenge with virus _-»J for 24 h ar 37°C the wells were washed with saline, stai¬ ned for 15 minutes with methylviolet and then washed with tap water. The 50% endpoints of the dose-response curves were determined visually in relation to cell and virus controls. In the assay system, one antiviral unit of the standard preparation corresponds with 150 pg of recom¬ binant rat IFN-y. EXAMPLE 6 Effects on skeletal muscle cell cultures Preparation of rat muscle cell cultures was carried out as described by [Kelic, S., Olsson, T. and Kristensson, K.J. Neurol.Sci. 1993, 114, 62]. The cells were grown either on collagen coated cover slips in 24 well multidish plates (Nunc, Roskilde, Denmark) or in microwell plates. Myobiast proliferation was determined according to Foster et al [Foster, R.F., Thompson, J.M. and Kaufman, S.J. Dev.Biol. 1987, 122, 11], Briefly the cells were ex¬ posed to the thymidine analogue bromodeoxyuridine (BrdUrd; Sigma) and incorporation of BrdUrd was visualized by incu¬ bating the cells with monoclonal FITC-conjugated anti- BrdUrd antibodies (Becton and Dickinson, San Jose, Ca., USA). To identify myoblasts the cultures were double-la¬ belled by adding a polyclonal anti-skeletal muscle desmin antibody (Sigma), which was visualized by a TRITC-conjuga- ted swine anti-rabbit IgG (Dakopatts, Copenhagen, Denmark). Proliferation index was determined as the number of BrdUrd positive myoblasts divided by the total number of myoblasts. The fusion index was defined as the number of myotube nuclei divided by the total number of nuclei of desmin positive cells [Honda, H. and Rostami, A. Proc.Natl.Acad.Sci. USA 1989, 86, 7007]. Binding of 125I-α-bungarotoxin (New England Nuclear, Boston, USA) to the cells was performed as described by Andre et al [Andre, P., Braun, S., Passaquin, A.C., Coupin, G., Bartholeyns, J., Warter, J.M. and Poindron, P.J. Neurosci.Res. 1988, 19, 297]. The equilibrium binding was performed with 1 pmol/ l of 125I-α-bungarotoxin at 37°C for 30 min. The cells were detached from the dishes with 1 ml 1M NaOH and bound radioactivity was measured in a gamma-counter. Specific binding was taken as the diffe¬ rence between binding in the absence and presence of 2 x excess of unlabelled cold cc-bungarotoxin (Sigma). To vi- sualize AChR distribution the method described by Schaffner and Daniels [Schaffner, A.E. and Daniels, M.P. J. eurosci. 1982, 2, 623] was used. The cultures were in¬ cubated with FITC-conjugated α-bungarotoxin (Sigma), 10 μg/ml, for 1 h, followed by extensive rinsing. The average number of ventral and dorsal receptor aggregates per yo- tube was determined in more than 60 myotubes in each of two cultures. All readings were done blindly under coded numbers. MHC antigen expression in skeletal muscle cell cultu¬ res was studied essentially as described above for macro¬ phages. Cell cultures grown in microwell plates and expo¬ sed to N-IFN-2T and recombinant IFN-y were fixed in forma¬ lin/picric acid and preincubated with 2% normal horse serum (Vector) at room temperature for 30 min, followed by incubation with monoclonal antibodies against MHC class I (0x18, Serotec, Oxford, England), 3 μg/ml, and MHC class II (0x6, Serotec),/3 μg/ml, overnight at +4°C. After rin¬ sing, the cells were incubated with biotinylated anti- mouse IgG (Vector), diluted 1/100, washed and incubated with avidin-biotin-alkaline-phosphate complex. The level of phosphatase enzyme activity measured as described above. For studies on myoblast proliferation and fusion, the cells were exposed 24 h after plating to purified N-IFN-2 or to recombinant IFN-tf at varying concentrations. The molecules were added every 24 h for 4 consecutive days and then taken for examinations. AChR aggregation, binding studies and MHC antigen expression studies were performed in cultures 6 days after plating and after a 24 h exposure to the factor. EXAMPLE 7 Results of experiments The remarkable feature of recombinant IFN-2 to act as a growth stimulus for T.b.b., as well as our observation in vitro indicating that N-IFN-y has a similar effect, was utilized for evaluation of the present purifications of N- IFN-Z. Thus, we first studied the T.b.b. growth stimulatory activity of a one-step antibody affinity purification of N-IFN-2, e.g. material eluted from the DBl antibody coup¬ led column. Data from one such experiment is shown in Fig. 1. Figure 1 shows effects of DBl column eluted N-IFN-y (diluted 10 " .10 ) and rIFN-y (1000 μ/ml) on trypanosome growth measured by counting numbers of parasites (A) or 3 H-thymidine incorporation 4 and 8 h after plating. 0 denotes cultures receiving no stimulus. Each staple rep¬ resents the mean value from 3 cultures. Thin bars denote S.D. Recombinant rat IFN-2, used as positive control, con¬ sistently stimulated growth of T.b.b. estimated both as 3 number of parasites present in the cultures and as H- thymidine incorporated 4 and 8 h after plating. N-IFN-2 eluted from the DBl column also stimulated T.b.b. growth with an effect down to a dilution of 10 -4, corresponding to an approximate protein concentration of 0.1-0.3 ng/ml medium. The additional DBl6 affinity column was then used for purifications of N-IFN-?. Figure 2 shows evaluation of N-IFN-2 purification 3 from trigeminal ganglion homogenates by measuring H- thymidine incorporation by T.b.b. 4 h after plating. Each bar denotes readings from triplicate cultures. S.D. is indicated. Negative control cultures received no stimulus (medium alone), positive control cultures received 1000 μ/ml of rIFN-2. Fractions from the DBl and DBl6 columns were applied to obtain a final dilution of 10 in the cultures. (A) denotes fractions from the DBl column after passing the trigeminal homogenate, but before alkaline elution. (B) denotes alkali eluted material. (C) denotes fractions obtained after washing the column with buffer, pH 7.4. Fraction B was added to the DBl6 column and the bioactivity of alkali eluted material is shown. As shown in Fig. 2, alkali eluted and neutralized N- IFN-2 from these consecutive DBl and DBl6 columns stimula¬ ted T.b.b. growth while other fractions were negative. This procedure was consistently repeated on 7 different experimental occasions. SDS-PAGE and silver staining of N-IFN-2 eluted either from the DBl column or the two consecutive columns dis¬ played three distinct bands of 66, 62 and 54 kD. This is clear from Figure 3 which shows SDS-PAGE and silver stai- ning of N-IFN-y obtained after consecutive elutions from the DBl and DBl6 columns. These bands were also seen after SDS-PAGE of purified N-IFN-Z followed by blotting to nit¬ rocellulose and im unostaining (Western blot) using DBl, DBl6 and a rabbit polyclonal anti-IFN-y antiserum (data not shown). The polyclonal antibody also bound to recombi¬ nant rat IFN-2 (500 units) run in parallel, between 14 and 25 kD. It revealed no staining in this region in the gels with the N-IFN-2 bands (data not shown). Native N-IFN-? was run also on PAGE, to study if the purified protein de- tected on gels was responsible for bioactivity or if these were due to other contaminating molecules not stained on gels and with other migrating properties. Here, a single silver stained band appeared and T.b.b. growth stimulatory activity was confined to the gel strip corresponding to this protein. This is illustrated in Figure 4, where N- IFN-2 eluted from the DBl6 column was subjected to PAGE under non-reducing conditions. A single silver stained band appeared (fraction 7). A longitudinal strip was cut out from this gel and subsequently divided into 17 pieces. Each piece was homogenized in 1 ml PBS and further diluted _2 10 as final concentration in trypanosome cultures eva- 3 luated for growth stimulation with H-thymidine incorpora¬ tion. Note that bioactivity is confined to fraction 7 con¬ taining N-IFN-2 visible on gel. We next evaluated the capacity of N-IFN-y to induce MHC class I and II antigens on macrophages and cells in cultures of skeletal muscle. Macrophages were induced to express significantly increased levels of MHC class I an¬ tigen in all of 7 different experiments, even at a dilu¬ tion of 10 " , corresponding to a protein concentration of approximately 0.1 ng/ l. N-IFN-2 also induced a signifi- cant expression of MHC class II antigen by macrophages in _3 2 out of 3 experiments. In this case 10 dilutions of N- IFN-2 were active. In addition, N-IFN-y significantly in¬ duced muscle cells to express MHC class I antigens (in 7 out of 7 experiments) and MHC class II (in 4 out of 6 ex- _3 periments), in both cases at dilutions down to 10 . This is illustrated in Figures 5 and 6. Figure 5 shows N-IFN-Ϊ induced MHC class I (A) and II (B) antigens on macrophages. Microtitre plate cultured macrophages were exposed to different dilutions of N-IFN- 2. Recombinant IFN-2 was used as positive and medium alone (0) as negative control. After application of MHC antigen specific antibodies, enzyme labelled immunoreagents, and colour reaction absorbance was used. Each bar denotes readings from 6-10 cultures. S.D. is shown (***p<0.001, **p<0.01). The data shown in A are from one representative experiment out of 7, all of which N- IFN-2 caused significant induction of MHC class I. The data shown in B are from one representative experiment in which MHC class II was significantly induced. Figure 6 shows N-IFn-2 induced MHC class I (A) and II (B) induction on skeletal muscle cells in the cell-ELISA system. Each staple represents readings from 6 cultures. Control denotes culture with medium alone, while other staples show results of different dilutions of N-IFN-2 (**p<0.01, ***p<0.001). The data shown in A are from one representative experiment out of 7, all of which N-IFN-2 caused significant induction of MHC class I. The data shown in B are from one representative experiment in which MHC class II was significantly induced. Recombinant IFN-2 (lOOμ/ml) as positive control significantly induced MHC class I and II antigens in all experiments (data not shown). Antiviral activity of N-IFN-y was titrated in bio- assay measuring inhibition of VSV cytopathic effects. On two different experimental occasions, down to 1/160 dilu¬ tions of the N-IFN-y preparation showed distinct antiviral effects, approximately corresponding to 100-300 antiviral units/μg N-IFN-y. Recombinant IFN-2 and N-IFN-y acted similarly with regard to other parameters assessed in the muscle cell cultures. Thus, both recombinant IFN-2 and N-IFN-y signi- ficantly enhanced formation of myotubes enclosed (Table I). This was caused by an increased myoblast proliferation as revealed in the BrdUrd labelling experiments (Fig. 7) and estimation of the fusion index. Figure 7 shows proli¬ feration of myoblasts as estimated by the BrdUrd test af- ter exposure to two dilutions of N-IFN-y and to 100 μ/ml of recombinant IFN-y. 0 denotes control cultures, not ex¬ posed to any of these factors (*p<0.01, **p<0.001). Myotubes show clustering of AChRs of two types, name¬ ly broad, 200-400 μm large, ventral patches and linear, 10-50 μm aggregations at the dorsal, free surface. These latter dorsal aggregates showed a significant decrease in number after exposure to both recombinant" IFN-2 and N-IFN- y, while no changes in number of ventral patches was seen. Equilibrium binding studies showed no changes in the le- vels of AChRs between N-IFN-2 or recombinant IFN-2 exposed cultures and controls. Thus, in the presence of recombi¬ nant IFN-2 (100 U/ml f Q or 24 h), the Bmβx was 6200 cpm/dish and K_ was 1.22 x 10~ M. In no exposed control cultures, the B was 7400 cpm/dish and apparent K_ was 1.41 x 10 -9 M. These experiments were reproducibly repeated three times. As described above we have here purified N-IFN-y to homogeneity and shown that it shares important bioactivi- ties with those of lymphocyte derived recombinant IFN-2. The use of a two-step affinity column procedure with, an¬ tibodies of different isotype and directed against spati¬ ally distinct epitopes of lymphocyte derived IFN-y resul- ted in a high degree of purity of N-IFN-y. Since both DBl and DBl6 bound N-IFN-2 in these columns, the immunological cross-reactivity between lymphocyte derived IFN-2 and N- IFN-y involves more than one epitope of the proteins. The isolated N-IFN-Z appeared as three distinct bands, 54, 62 and 66 kD, after SDS-PAGE and silver stai¬ ning. These could represent breakdown products of one and the same protein or one protein with different states of glycosylation. The molecular size of the presently puri- fied N-IFN-2 differs from that of lymphocyte derived rat IFN-2, which is a 156 amino acid glycoprotein dimere with the monomer MW ranging between 14 and 25 kD due to diffe¬ rential glycosylation [van der Meide, P.H., Dubbeld, M., Vijverberg, K., Kos, T., Schellekens, H.J. Gen.Virol. 1986, 67, 1059; van der Meide, P.H., Borman, A.H., Beijaars, H.G., Dubbeld, M.A., Botman, C.A.D. and Schellekens, H. Lymphokine Res. 1989, 8, 439]. That N-IFN- 2f and lymphocyte derived IFN-y differs, is also indicated by our, and others, failure to detect mRNA for lymphocyte derived IFN-2 using Northern blotting with probes for IFN- 2 or synthetic oligonucleotide probes for the cytokine, a procedure which readily detects such mRNA in activated lymphocytes. Table 1: Effects of N-IFN- T and recombinant IFN- T on myotube formation and AChR aggregation a) Values are means 1SD of 7-10 randomly selected microscopic fields (0.13 mm ) or of, b) 60-70 counted myotubes. *:p<0.001, on comparison with cultures receiving no additive, Student's t-test.

Claims: CLAIMS 1. An interferon glycoprotein of neuronal origin N- IFN-2) which has a molecular weight of from about 50 to about 70 kDa, is capable of interaction with at least two monoclonals directed against spatially distinct epitopes of lymphocyte-derived IFN-2, exerts parasite-stimulating and anti-viral actions,, and is capable of induction of MHC and of enhancing muscle cell proliferation. 2. A glycoprotein according to claim 1, wherein said monoclonals are DBl and DBl6. 3. A glycoprotein according to claim 1 or 2, which on SDS-PAGE and silver staining displays three distinct bands corresponding to approximate molecular weights of 66, 62 and 54 kDa. 4. A pharmaceutical composition comprising an effec¬ tive amount of the glycoprotein of claim 1, 2 or 3 in admixture with a pharmaceutically acceptable vehicle or carrier. 5. A process for preparing an interferon glycoprotein of neuronal origin (N-IFN-2) which has a molecular weight of from about 50 to about 70 kDa, is capable of inter¬ action with at least two monoclonals directed against spatially distinct epitopes of lymphocyte-derived IFN-2T, exerts parasite-stimulating and anti-viral actions, and is capable of induction of MHC and of enhancing muscle cell proliferation, comprising the steps: a) providing a starting material originating from neurons of peripheral sensory ganglia; b) preparing a homogenate in suspension of the mate- rial from step a); c) passing the suspension resulting from step b) through a column having immobilized therein a first mouse monoclonal anti-rat IFN-y antibody and eluting the desired material using an alkaline buffer solution; d) passing the eluate from step c) through another column having immobilized therein a second mouse monoclo¬ nal anti-rat IFN-y antibody and repeating the elution pro- cedure of step c); and e) recovering the eluate from step d) comprising the desired glycoprotein. 6. A process according to claim 5, wherein said anti- bodies are DBl and DB16. 7. A process according to claim 5 or 6, wherein the elution procedure of steps c) and d) is performed with a buffer solution having a pH of about 12. 8. A method of performing interferon therapy on a mammal including man, comprising administering a therapeu¬ tically effective amount of the glycoprotein of claim 1, 2 or 3 or of the composition of claim 4. 9. A method according to claim 8, wherein said thera¬ py resides in the treatment of a viral infection, in the provision of cell growth regulation, or in the regulation of the production of a cell-produced substance. 10. A method of performing antiviral therapy to a mammal including man, comprising administering a viral infection inhibiting amount of the polypeptide of claim 1, 2 or 3 or the composition of claim 4 to said mammal.

Inventors: EDLUND CONNY, HOEJEBERG BO, KRISTENSSON KRISTER, LJUNGDAHL AAKE, OLSSON TOMAS, EDLUND, CONNY, HOEJEBERG, BO, KRISTENSSON, KRISTER, LJUNGDAHL, AAKE, OLSSON, TOMAS
Applicants: EDLUND CONNY, HOEJEBERG BO, KRISTENSSON KRISTER, LJUNGDAHL AAKE, OLSSON TOMAS, EDLUND, CONNY, HOEJEBERG, BO, KRISTENSSON, KRISTER, LJUNGDAHL, AAKE, OLSSON, TOMAS
Application year: 1994
Main classification: C07K14
IPC codes: A61K38, C07K14